We have developed a model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic (hyporesponsive) T cells. We call this state adaptive tolerance. We inject CD4+, cytochrome c-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse (called mPCC) expressing the cytochrome c antigen under the control of the MHC class I promoter and an immunoglobin heavy chain enhancer. Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In this adaptive tolerant state, cytokine responses to high doses of antigen in vitro are inhibited 90%. Expression on restimulation of early activation markers, such as CD69 and CD25, is also impaired, and recent biochemical studies of signal transduction show an inhibition of Zap70 activation and calcium mobilization following T cell receptor engagement. In vivo Budr labeling showed a slow T cell turnover of about 5% per day. This hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. Interestingly, if the retransfer is into a host expressing the antigen, the cells remain hyporesponsive and slowly decrease their IL-2 and IFN gamma production by another 6-10 fold over 3-4 weeks. This deeper state of anergy suggested that the tolerance process is adaptable to different levels. To test this idea more directly, we studied a second transgenic mouse (ePCC) expressing about a 4 fold lower amount of antigen. TCR transgenic CD4 T cells injected into this mouse also proliferated and entered into an anergic state, although with slower kinetics. Eventually, however, the turn over rate in vivo was comparable to that of T cells in the mPCC environment. Interestingly, in vivo restimulation on retransfer into a fresh mPCC mouse revealed a greater impairment in the proliferative ability of T cells resident in a higher antigen presentation environment. We also observed subtle differences in TCR signaling and in vitro cytokine production consistent with differential adaptation. Notably, the system failed to similarly compensate to the persistent stimulus in vivo at the level of CD69 expression and actin polymerization. The greater responsiveness of T cells residing in a host with a lower level of antigen presentation allowed us to demonstrate for the first time an intrinsic "tuning" process in mature T lymphocytes, although one more complex than current theoretical models would have predicted. During the past year we have explored the role of the inhibitory receptor, CTLA-4, in the adaptive tolerance process. Surprisingly, we found that the tuning process could be induced and the hyporesponsive state maintained in the absence of CTLA-4. This also included induction into the deeper tolerant state. Instead CTLA-4 inhibited late T cell expansion in vivo and curtailed IL-2 and IFN-gamma production. Removal of this braking function in the CTLA-4 deficient mice may explain their lymphoproliferative dysregulation. In an independent study in the lab on the role of DNA demethylation in transcriptional regulation of the IL-2 gene, we located a small 600 bp region in the promoter/enhancer of the gene that demethylates in T lymphocytes following activation, and remains demethylated thereafter. This epigenetic change was necessary and sufficient to enhance transcription in reporter plasmids. The demethylation process started as early as 20 minutes after stimulation in vivo and was not prevented by a G1 to S phase cell cycle inhibitor (Rapamycin) that blocks DNA replication. These results imply that the demethylation process proceeds by an active enzymatic mechanism and suggests the existence of a site-specific demethylase. We speculate that the major function of this event is to enhance the amount or rate of transcription of the IL-2 gene in a memory T cell population to facilitate the rapid production of this cytokine in a secondary immune response. During the past year we have turned our attention to another cytokine gene, IFN-gamma. Previous reports had suggested that the promoter region of this gene was also methylated on CpGs in naive T cells. Our studies in the mouse, however, demonstrated that a 300 bp region of the IFN-gamma promoter is hypomethylated in both CD4 and CD8 naive T cells. This state was also present in thymocytes as early as the double negative stage. Following activation of the naive T cells under Th2 conditions (adding IL-4 and blocking IFN-gamma and IL-12), this promoter region became hypermethylated and the cells failed to produce IFN-gamma on reactivation. Remethylation was far less prominent when the cells were differentiated under Th1 conditions (blocking IL-4 and adding IL-12 and IFN-gamma), where the cells produced lots of IFN-gamma on reactivation. While more studies are needed to explore the relationship between promoter methylation and T cell IFN-gamma cytokine production, our experiments support a new model for IFN-gamma chromatin structural changes in murine T cell development. Why the DNA methylation status of the IL-2 and IFN-gamma genes in naive T cells should be different is the current focus of research in the laboratory.